Post-treatment Flare-up Incidence after Using Nano Zinc Oxide Eugenol Sealer in Mandibular First Molars with Irreversible Pulpitis

Statement of the Problem: Some patients may report moderate-to-severe pain and/or swelling following root canal treatment, which is undesirable for both the patient and dentist and may require an unscheduled emergency visit by patients to relieve their symptoms. Purpose: The aim of this study was to evaluate the post-treatment flare-up incidence following application of nano zinc oxide-eugenol (NZOE) sealer in mandibular first molars with irreversible pulpitis. Materials and Method: This single-blinded study was performed on 60 patients having mandibular first molars with irreversible pulpitis. After signing the written consent form, the patients were randomly divided into two groups considering their age range (20-34 and 35-50 years). Individuals without systemic diseases and with a ﬁrst mandibular molar diagnosed with irreversible pulpitis due to caries, no sinus track and abscess, normal periapical radiographic appearance, no spontaneous pre-treatment pain, not taken any medication for at least 8 hours before the treatment visit were included in this study. Patients of both sexes with age range of 20 to 50 years were selected. In order to obturate the root canal space, AH26 sealer was used in the one group and the synthesized NZOE was applied in another group. The patients were then given questionnaires to report the severity of pain and presence of swelling during 6, 18, 24, and 48 hours of follow-up. The data was analyzed using SPSS v.19 software applying repeated measures ANOVA. The significance level was set at 0.05. Results: The severity of pain was significantly lower in NZOE group at 24 hours post-treatment (p= 0.003). Patients reported no symptoms of swelling in any group. Conclusion: NZOE sealer manifested satisfactory results and could be regarded as a promising substitute to routine sealers since patients may experience less pain during the first hours after receiving root canal therapy.


Introduction
The main objectives of root canal therapy are to achieve long-term comfort, function, and aesthetics for the patients and prevention of reinfection of tooth. These objectives are provided through complete cleaning, shaping, and obturation of canals of affected teeth [1]. However, some patients may report moderate-to-severe pain and/or swelling following root canal treatment that is detrimental for both patient and dentist and may entail an unscheduled emergency visit by patients to relieve their symptoms. The rate of flare-up following root canal therapy (RCT) has been reported to range from 2.53% to 58% [2]. Pack et al. reported 40% of the patients had post-operative pain 24 hours after RCT and this decreased to 11% after one week [3]. Although the exact reasons for flare-up are not clearly understood, it is accepted that flare-up is a multi-factorial phenomenon and there is no single reason for its occurrence [4]. Microbiological, chemical, mechanical, and host variables can induce pain and swelling following root canal treatment [5]. In addition, history of preoperative pain, gender and age factors, the pre-operative pulp status, the type of tooth, the type of treatment (initial or retreatment), and a history of allergy may all influence the rate of flare-up [6]. In addition, some studies have demonstrated the effect of several factors such as instrumentation technique, type of analgesic, method of analgesic prescription, type of rotary instrument, anesthetic solution, occlusal reduction, and method of root canal filing on incidence of flare up [7][8].
In obturation of the root canal system, sealer is used to prevent penetration of microorganisms and their byproducts. In the other hand, sealer is in direct contact with periapical tissues and may cause inflammation, tissue degeneration, and delay in wound healing. Therefore, the ideal root canal sealer should be non-cytotoxic, non-mutagenic, and immunologically compatible with periapical tissues [9]. Currently, a large variety of sealers with different formulas and physical properties are available for use. However, they all have their limitations. It is difficult to produce a sealer with proper physicochemical properties while being biocompatible for long-term. For many years, zinc oxide-eugenol (ZOE)based sealers have been widely used in endodontic practice. The use of nanotechnology has allowed many developments in dentistry and advances in oral-healthrelated nano material and therapeutic methods [10].
Some of the advantages of using nano particles in endodontic sealers include improving their physicochemical characteristics, enhancing the antibacterial property, better penetration into the dental tubules, decreasing microleakage, and increasing biocompatibility [11][12].
It has been shown that incorporating zinc oxide nano particles enhances the physicochemical characteristics (setting time, flow, solubility, dimensional stability, and radiopacity) of Grossman sealer [12]. Kesler Shvero et al. [13] demonstrated that epoxy resin-based surfaces with cationic nano particles attracted and sacrificed Enterococcus faecalis. DaSilva et al. [14] showed that incorporating chitosan nano particles into ZOE sealer reduced the formation of biofilm within the sealerdentin interface. In addition, it has been reported that nano-ceramic sealer had better cytocompatibility compared to Endoseal MTA considering the effects on cell spreading and proliferation [15].
Recently, a new endodontic sealer with nano-sized ZO powder particles (NZOE) has been developed in the Dental Material Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. This sealer is similar to various ZOE-based sealers, but with different sizes of ZOE nanoparticles. In previously published articles, we verified that NZOE sealer had less microleakage and better antibacterial property in comparison with Pulpdent and AH-26 sealers [16]. In an animal study, it was observed that the histocompatibility properties of NZOE were comparable to the above-mentioned commercial sealers [17]. Moreover, the cytotoxicity of NZOE on murine L929 cell line was comparable to that of Pulpdent and was lower than AH-26 sealer [18]. In another study, the cytotoxicity of NZOE sealer on human gingival fibroblasts isolated from healthy subjects was evaluated and NZOE exhibited lower cytotoxicity compared to Pulpdent and AH-26 [19].
ZOE -based sealers are the oldest sealer used in endodontic therapy. Zinc oxide (ZO) is a valuable component of these sealers that is very effective as an antimicrobial agent. Many reforms have been done on these sealers in order to improve their properties and many commercial models are available [20]. In addition, ZOE-based cements have been found to possess favorable characteristics in terms of biocompatibility [19][20].
To the best of our knowledge, the use of nanostructured materials as sealers in root canal therapy is limited to two or three types of nano-structured hydroxyapatite alone or in combination with epoxy resin (Nanoseal) [20][21][22]. and NZOE as a sealer in mandibular molars with irreversible pulpitis that were treated in one visit.

Materials and Method
The Ethics Committee of Mashhad University of Medical Sciences (Ir.mums.dentistry.rec.1397.044 and Iranian Registry of Clinical Trials ID No. IRCT2018071136 542) approved the protocol of this study.

Synthesis of ZnO nano-particles
In this work, ZnO nano-powders were prepared by a modified sol-gel method, using gelatin [23].
To prepare 5 g of the final product, first a solution of gelatin (type B from bovine skin, Sigma Aldrich) was prepared by dissolving 10 g of gelatin in 150 mL of deionized water at 60°C. Then, appropriate amounts of zinc nitrate (Zn (NO 3 ) 2 .6H 2 O; Merck %99) were dissolved in minimum deionized water at room temperature. Then, the two solutions were mixed and stirred for 8 hours while the temperature was kept at 80°C. Finally, the pure resins were calcined at different temperatures of 500, 600, and 700°C to obtain ZnO nano-powders.
Morphological and structural properties of the prepared ZnO were characterized by x-ray diffraction (XRD) and transmission electron microscopy (TEM) techniques.

Procedural steps
The sample size was calculated based on an alpha of 0.05 with power of 80%, indicating that 27 patients would be required in each group but after considering the dropout rate of 10%, the sample size increase to 30 patients in each group.
Inclusion criteria were as follows: individuals without systemic diseases and with a first mandibular molar diagnosed with irreversible pulpitis due to caries, no sinus track and abscess, normal periapical radiographic appearance, no spontaneous pre-treatment pain, not having taken any medication for at least 8 h before the treatment visit. Patients of both genders ranging from 20 to 50 years of age were selected. During the root canal procedure, teeth that could not be treated in a single visit or patients who interrupted treatment were excluded. In addition, the patient with severe periodontal disease, over instrumentation or overfilling beyond the root canal space, teeth that could not be isolated with rubber dam, root canal calcification, root resorption and teeth that were not suitable for further restoration were excluded.
All the treatment procedures were performed by a postgraduate student in the Endodontic Department, Mashhad Dental School, Iran, from May to August 2018. A total of 60 teeth were randomly assigned to two groups using a random-digit table. Before initiating treatment, the patients were asked to rate their pretreatment pain on a visual analogue scale from 0 to 9.  In the present study, for prevention of possible bias, patients with similar preoperative conditions were included. As the preoperative pain would have a significant effect on postoperative pain [24], only patients with no spontaneous pain were included. In addition, because pre-treatment analgesic consumption would influence the postoperative pain following root canal treatment [25], all patients were asked about the medication they used. The participants should not have taken any medication for at least 8 h before the treatment visit. According to systematic reviews, single-visit root canal treatment has no significant difference with multi-visit treatment regarding postoperative pain [26].

Based on the inclusion and exclusion criteria, a total of
Therefore, in the present study, single-visit root canal treatment was undertaken. In this study, the distribution of age and gender were the same for both groups due to the established association of these variables to endodontic flare-up. All the treatments carried on mandibular first molars with irreversible pulpitis and instrumentation of the root canals was completed in a crown-down technique using rotary nickel titanium files as it has shown to be associated with less debris and irrigants extrusion apically, which has a clinical implication in decreasing the incidence of postoperative pain [27]. In addition, attempt was done to avoid over-instrumentation of root canal during filing. Therefore, both experimental groups were identical in terms of age, gender, tooth type, preoperative pain intensity, irrigation solution, instrumentation, and obturation technique. The results of a systematic review showed that during the first 48 h after root canal treatment, post-operative pain decreased significantly [28]. Therefore, we limited our research to the first 48 h after treatment.
Various methods were used for evaluating the effect of different variables on postoperative endodontic pain.  In the present study, a VAS was used to compare postoperative pain intensity because it is a simple tool with high reliability and validity and more sensitive to small changes than descriptive ordinal scales [8].
There are some studies on the effect of irrigation type [29] and irrigation concentrations [30]  Over-instrumentation, excessive amount of sealer, the excessive compaction force, hydrostatic pressure, the use of lentulo spirals, immature canal apices, and root tip resorption can increase the risk of sealer extrusion [32].
In this study, the most post-operative pain was reported by AH 26 group during the 24 h after treatment.
This can be due to mild-to-moderate irritating effects of AH26 when freshly prepared and the toxicity is due to the release of a small amount of formaldehyde, epoxide bisphenol resin, or amines during the chemical setting process [33]. In addition, Ruparel et al. [34] revealed that resin-based sealer can directly activate trigeminal nociceptors and leading to release of CGRP and may therefore cause pain and neurogenic inflammation. In another study, Huang et al. [35] reported that AH26 sealer had significant cytotoxicity in the periradicular tissues by inducing inflammatory mediators such as receptor activator of nuclear factor kappa B ligand, nitric oxide synthase, cyclooxygenase-2, and reactive oxygen species. The direct activation along with the immunologic response may underlie flare-up occurrences. On the other hand, two in vitro studies reported that the cytotoxicity of NZOE on murine L929 cell line [18] and on human gingival fibroblasts [19] was lower than AH-26 sealer.
According to the result of the present study and considering the lesser microleakage and better antibacterial property of NZOE in comparison to AH-26 sealers [16] and concerning the lower cytotoxicity of NZOE than AH-26 sealer [17][18][19], NZOE sealer could be regarded as endodontic sealer for root canal obturation.

Conclusion
Up to 24 h following root canal treatment, the use of NZOE sealer was associated with significantly lesser pain than the use of AH26 in studied patients.

Acknowledgments
This study was supported by a grant from the Vice

Conflicts of Interest
None declared